The HIV/SIV accessory protein negative factor (Nef) is a key determinant of high levels of viremia and the progression to AIDS. It is expressed early and abundantly in the viral replicative cycle. Being a myristylated, membrane associated protein, it also leads to rapid internalization of CD4 and sequestration of MHC I determinants, cellular activation and increased infectivity of progeny virions. This proposal aims to dissect the mechanism of action of Nef from HIV in great detail. First, the role of the vacuolar ATPase in the trafficking of Nef, CD4 and MHC I determinants will be examined. This interaction could also affect the processing of viral polyprotein precursors, the maturation of progeny virions as well as their uncoating in infected cells. Biochemical, genetic, virological and pharmacological approaches will be used to dissect the role of V-ATPase in viIa1 replication. To reverse the orientation of V-ATPase in the core, Nef will also be targeted into virions as a fusion protein with Vpr. Nef also affects cellular signaling cascades. Tyrosine and serine threonine kinases participate in this process. Our attention is focused on the phophospoinositol 3 kinase, the guanine nucleotide exchange factor Vav, small GTPases Cdc42 and Racl, and p21 activated kinases, PAK1 and 2. Together, they form the signalosome on Nef that leads to cytoskeletal rearrangements and downstream effector functions. This signaling cascade will be dissected in greater detail, with emphasis on additional players, effects on the cytoskeleton, the formation of lipid rafts and viral production. For example, do actin polymerization and lipid raft aggregation affect the fitness of HIV and its subsequent infectivity? Finally, the role of lipid rafts in viral replication will be investigated in great detail. First, the binding of multimers of Nef to the GagPol precursor wil1 be investigated. Then, the presence of these megacomplexes in these detergent resistant membranes will be followed biochemically and morphologically. Different targeting of Nef will be used to divert the formation of new virions away from lipid rafts. Moreover, properties of an unusual allele of Nef, from HIV-1/F12 will be investigated for its dominant negative properties in viral replication.